Abstract

The G12/13 class of heterotrimeric G proteins is unique in its ability to stimulate cellular transformation via overexpression of the wildtype α subunit, Gα12 or Gα13, absent of activating mutations. We have studied the mechanism of wildtype Gα12/13 signaling to the transcriptional activator Serum Response Factor (SRF), a pathway implicated in multiple cancer types. When overexpressed in cultured human embryonic kidney cells, wildtype Gα13 showed robust stimulation of this transcriptional response. This signaling by Gα13 was blunted by overexpression of G protein β1 and γ2 subunits, suggesting that aberrant signaling by the overexpressed α subunit results from stoichiometric imbalance between the heterotrimeric G protein subunits. Next, using an epitope-tagged Gα13 to track its subcellular location, we discovered the overexpressed α subunit shifted from a membrane-associated fraction to a soluble fraction, coincident with its sharp increase in SRF signaling. Overexpression of the β1γ2 dimer caused re- localization of wildtype Gα13 to the membrane-associated fraction, coincident with its diminished signaling. Interestingly, our preliminary data utilizing a non-prenylated γ2 subunit suggest the ability of the βγ dimer to suppress signaling by overexpressed, wildtype Gα13 is independent of βγ association with the cell membrane. We currently are examining effects of this non-membrane bound dimer on subcellular localization of wildtype Gα13. Furthermore, to characterize protein interactions specific to overexpressed Gα13, we have constructed Glutathione-S-Transferase (GST) fusions of several proteins reported as specific targets of Gα13, including RGS16, PYK2, and Talin-1. The role of Gα13 activation in binding these effector proteins will be examined by using aluminum, magnesium, and fluoride ions to chemically activate the α subunit.

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